The distribution and interactions of molecules in three-dimensionally organized cellular networks are fundamen- tal to the function of living systems. However, to date, a complete understanding of how local molecular mecha- nisms are integrated over larger scales to support tissue functions, or contribute to disease initiation, is still lacking. The challenges are mainly due to the limitations in imaging technology to provide molecular specificity, nanometer-scale resolution, ultrafast speed across larger volumes of tissue. To address the challenge, the pro- posed research program investigates the physical and engineering principles underlying optical imaging in com- plex biological materials, and utilizes these principles to develop new biophotonic tools for next-generation light microscopy. The objective of this proposal is to establish a research program on transformative bioimaging tech- nology for high-throughput extraction of single-molecule information in biological systems. Specifically, the pro- posed research program proceeds in three major directions to develop and apply enabling technologies for un- derstanding the ultrastructural architecture, fast dynamics, and spatiotemporal-multiplexed molecular information in complex biological systems: 1) Wavefront-engineered super-resolution microscopy to allow nanometer-scale imaging at and beyond the tis- sue level with isotropic 3D resolution and large imaging depth; 2) High-resolution light-field microscopy and computational super-resolution imaging to enable ultrafast, live im- aging of large-scale, volumetric biological dynamics and activities; and 3) Spatiotemporal-multiplexed imaging and a proof-of-principle investigation of the brain immune system. The research integrates and translates innovations in physical concepts, computational frameworks, and ad- vanced optical engineering and instrumentation into enabling technologies for biomedical investigations. The significant impact of the work will advance the imaging power across unexplored regimes in both space and time for a better understanding of the molecular basis for the functions of tissues and organisms. The spatiotemporal- multiplexed imaging of the brain immune system will lay the technological foundation for future systematic inves- tigations of the role of microglia in brain homeostasis, circuit formation, and disease initiation and protection. In the long term, the proposed program is expected to not only provide new insights for brain study, but also open up many new pathways to a broad range of biomedical research, and ultimately enable new discoveries to ad- dress challenges in human well-being.